Protein Tyrosine Phosphatases: A new paradigm in an old signaling system?

Protein Tyrosine Phosphatases reverse cellular signals initiated by growth factors receptors and other tyrosine kinases by dephosphorylating phosphotyrosine on target proteins. The activity of these enzymes is crucial for maintaining cell homeostasis, yet these enzymes have been often dismissed as humble house-keeping proteins. Understandably, mutations and changes in expression patterns of Protein Tyrosine Phosphatases are implicated in tumorigenesis and various carcinomas. The conserved nature of their catalytic domains makes drug discovery a challenging pursuit. In this review, we focus on describing the various classes of Protein Tyrosine Phosphatases and their catalytic domains. We also summarize their role in cancer and neurodegenerative diseases using specific members as the model system. Finally, we explain the dichotomy in the biological role of catalytically active vs the pseudoenzyme forms of Protein Tyrosine Phosphatases in the context of their membrane bound receptor forms. This chapter aims to provide a current understanding of these proteins, in the background of their foundational past research.

Two PKA RIα holoenzyme states define ATP as an isoform-specific orthosteric inhibitor that competes with the allosteric activator, cAMP.

Protein kinase A (PKA) holoenzyme, comprised of a cAMP-binding regulatory (R)-subunit dimer and 2 catalytic (C)-subunits, is the master switch for cAMP-mediated signaling. Of the 4 R-subunits (RIα, RIß, RIIα, RIIß), RIα is most essential for regulating PKA activity in cells. Our 2 RIα2C2 holoenzyme states, which show different conformations with and without ATP, reveal how ATP/Mg2+ functions as a negative orthosteric modulator. Biochemical studies demonstrate how the removal of ATP primes the holoenzyme for cAMP-mediated activation. The opposing competition between ATP/cAMP is unique to RIα. In RIIß, ATP serves as a substrate and facilitates cAMP-activation. The isoform-specific RI-holoenzyme dimer interface mediated by N3A-N3A' motifs defines multidomain cross-talk and an allosteric network that creates competing roles for ATP and cAMP. Comparisons to the RIIß holoenzyme demonstrate isoform-specific holoenzyme interfaces and highlights distinct allosteric mechanisms for activation in addition to the structural diversity of the isoforms.

Dynamic allostery-based molecular workings of kinase:peptide complexes.

A dense interplay between structure and dynamics underlies the working of proteins, especially enzymes. Protein kinases are molecular switches that are optimized for their regulation rather than catalytic turnover rates. Using long-simulations dynamic allostery analysis, this study describes an exploration of the dynamic kinase:peptide complex. We have used protein kinase A (PKA) as a model system as a generic prototype of the protein kinase superfamily of signaling enzymes. Our results explain the role of dynamic coupling of active-site residues that must work in coherence to provide for a successful activation or inhibition response from the kinase. Amino acid networks-based community analysis allows us to ponder the conformational entropy of the kinase:nucleotide:peptide ternary complex. We use a combination of 7 peptides that include 3 types of PKA-binding partners: Substrates, products, and inhibitors. The substrate peptides provide for dynamic insights into the enzyme:substrate complex, while the product phospho-peptide allows for accessing modes of enzyme:product release. Mapping of allosteric communities onto the PKA structure allows us to locate the more unvarying and flexible dynamic regions of the kinase. These distributions, when correlated with the structural elements of the kinase core, allow for a detailed exploration of key dynamics-based signatures that could affect peptide recognition and binding at the kinase active site. These studies provide a unique dynamic allostery-based perspective to kinase:peptide complexes that have previously been explored only in a structural or thermodynamic context.

Tuning the "violin" of protein kinases: The role of dynamics-based allostery.

The intricacies of allosteric regulation of protein kinases continue to engage the research community. Allostery, or control from a distance, is seen as a fundamental biomolecular mechanism for proteins. From the traditional methods of conformational selection and induced fit, the field has grown to include the role of protein motions in defining a dynamics-based allosteric approach. Harnessing of these continuous motions in the protein to exert allosteric effects can be defined by a "violin" model that focuses on distributions of protein vibrations as opposed to concerted pathways. According to this model, binding of an allosteric modifier causes global redistribution of dynamics in the protein kinase domain that leads to changes in its catalytic properties. This model is consistent with the "entropy-driven allostery" mechanism proposed by Cooper and Dryden in 1984 and does not require, but does not exclude, any major structural changes. We provide an overview of practical implementation of the violin model and how it stands amidst the other known models of protein allostery. Protein kinases have been described as the biomolecules of interest. © 2019 IUBMB Life, 71(6):685-696, 2019.

Globally correlated conformational entropy underlies positive and negative cooperativity in a kinase's enzymatic cycle.

Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. For signaling enzymes, substrate recognition, binding, and product release are often rate-determining steps in which enthalpy-entropy compensation plays a crucial role. While the nature of enthalpic interactions can be inferred from structural data, the molecular origin and role of entropy in enzyme catalysis remains poorly understood. Using thermocalorimetry, NMR, and MD simulations, we studied the conformational landscape of the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous phosphoryl transferase involved in a myriad of cellular processes. Along the enzymatic cycle, the kinase exhibits positive and negative cooperativity for substrate and nucleotide binding and product release. We found that globally coordinated changes of conformational entropy activated by ligand binding, together with synchronous and asynchronous breathing motions of the enzyme, underlie allosteric cooperativity along the kinase's cycle.

A Catalytically Disabled Double Mutant of Src Tyrosine Kinase Can Be Stabilized into an Active-Like Conformation.

Tyrosine kinases are enzymes playing a critical role in cellular signaling. Molecular dynamics umbrella sampling potential of mean force computations are used to quantify the impact of activating and inactivating mutations of c-Src kinase. The potential of mean force computations predict that a specific double mutant can stabilize c-Src kinase into an active-like conformation while disabling the binding of ATP in the catalytic active site. The active-like conformational equilibrium of this catalytically dead kinase is affected by a hydrophobic unit that connects to the hydrophobic spine network via the C-helix. The αC-helix plays a crucial role in integrating the hydrophobic residues, making it a hub for allosteric regulation of kinase activity and the active conformation. The computational free-energy landscapes reported here illustrate novel design principles focusing on the important role of the hydrophobic spines. The relative stability of the spines could be exploited in future efforts to artificially engineer active-like but catalytically dead forms of protein kinases.

A dynamic hydrophobic core orchestrates allostery in protein kinases.

Eukaryotic protein kinases (EPKs) constitute a class of allosteric switches that mediate a myriad of signaling events. It has been postulated that EPKs' active and inactive states depend on the structural architecture of their hydrophobic cores, organized around two highly conserved spines: C-spine and R-spine. How the spines orchestrate the transition of the enzyme between catalytically uncommitted and committed states remains elusive. Using relaxation dispersion nuclear magnetic resonance spectroscopy, we found that the hydrophobic core of the catalytic subunit of protein kinase A, a prototypical and ubiquitous EPK, moves synchronously to poise the C subunit for catalysis in response to binding adenosine 5'-triphosphate. In addition to completing the C-spine, the adenine ring fuses the ß structures of the N-lobe and the C-lobe. Additional residues that bridge the two spines (I150 and V104) are revealed as part of the correlated hydrophobic network; their importance was validated by mutagenesis, which led to inactivation. Because the hydrophobic architecture of the catalytic core is conserved throughout the EPK superfamily, the present study suggests a universal mechanism for dynamically driven allosteric activation of kinases mediated by coordinated signal transmission through ordered motifs in their hydrophobic cores.

Mutation of a kinase allosteric node uncouples dynamics linked to phosphotransfer.

The expertise of protein kinases lies in their dynamic structure, wherein they are able to modulate cellular signaling by their phosphotransferase activity. Only a few hundreds of protein kinases regulate key processes in human cells, and protein kinases play a pivotal role in health and disease. The present study dwells on understanding the working of the protein kinase-molecular switch as an allosteric network of "communities" composed of congruently dynamic residues that make up the protein kinase core. Girvan-Newman algorithm-based community maps of the kinase domain of cAMP-dependent protein kinase A allow for a molecular explanation for the role of protein conformational entropy in its catalytic cycle. The community map of a mutant, Y204A, is analyzed vis-à-vis the wild-type protein to study the perturbations in its dynamic profile such that it interferes with transfer of the γ-phosphate to a protein substrate. Conventional biochemical measurements are used to ascertain the effect of these dynamic perturbations on the kinetic profiles of both proteins. These studies pave the way for understanding how mutations far from the kinase active site can alter its dynamic properties and catalytic function even when major structural perturbations are not obvious from static crystal structures.

Decoding the Interactions Regulating the Active State Mechanics of Eukaryotic Protein Kinases.

Eukaryotic protein kinases regulate most cellular functions by phosphorylating targeted protein substrates through a highly conserved catalytic core. In the active state, the catalytic core oscillates between open, intermediate, and closed conformations. Currently, the intramolecular interactions that regulate the active state mechanics are not well understood. Here, using cAMP-dependent protein kinase as a representative model coupled with biochemical, biophysical, and computational techniques, we define a set of highly conserved electrostatic and hydrophobic interactions working harmoniously to regulate these mechanics. These include the previously identified salt bridge between a lysine from the ß3-strand and a glutamate from the αC-helix as well as an electrostatic interaction between the phosphorylated activation loop and αC-helix and an ensemble of hydrophobic residues of the Regulatory spine and Shell. Moreover, for over three decades it was thought that the highly conserved ß3-lysine was essential for phosphoryl transfer, but our findings show that the ß3-lysine is not required for phosphoryl transfer but is essential for the active state mechanics.

Divalent Metal Ions Mg²âº and Ca²âº Have Distinct Effects on Protein Kinase A Activity and Regulation.

cAMP-dependent protein kinase (PKA) is regulated primarily in response to physiological signals while nucleotides and metals may provide fine-tuning. PKA can use different metal ions for phosphoryl transfer, yet some, like Ca(2+), do not support steady-state catalysis. Fluorescence Polarization (FP) and Surface Plasmon Resonance (SPR) were used to study inhibitor and substrate interactions with PKA. The data illustrate how metals can act differentially as a result of their inherent coordination properties. We found that Ca(2+), in contrast to Mg(2+), does not induce high-affinity binding of PKA to pseudosubstrate inhibitors. However, Ca(2+) works in a single turnover mode to allow for phosphoryl-transfer. Using a novel SPR approach, we were able to directly monitor the interaction of PKA with a substrate in the presence of Mg(2+)ATP. This allows us to depict the entire kinase reaction including complex formation as well as release of the phosphorylated substrate. In contrast to Mg(2+), Ca(2+) apparently slows down the enzymatic reaction. A focus on individual reaction steps revealed that Ca(2+) is not as efficient as Mg(2+) in stabilizing the enzyme:substrate complex. The opposite holds true for product dissociation where Mg(2+) easily releases the phospho-substrate while Ca(2+) traps both reaction products at the active site. This explains the low steady-state activity in the presence of Ca(2+). Furthermore, Ca(2+) is able to modulate kinase activity as well as inhibitor binding even in the presence of Mg(2+). We therefore hypothesize that the physiological metal ions Mg(2+) and Ca(2+) both play a role in kinase activity and regulation. Since PKA is localized close to calcium channels and may render PKA activity susceptible to Ca(2+), our data provide a possible mechanism for novel crosstalk between cAMP and calcium signaling.

Single Turnover Autophosphorylation Cycle of the PKA RIIß Holoenzyme.

To provide tight spatiotemporal signaling control, the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) holoenzyme typically nucleates a macromolecular complex or a "PKA signalosome." Using the RIIß holoenzyme as a prototype, we show how autophosphorylation/dephosphorylation of the RIIß subunit, as well as cAMP and metal ions, contribute to the dynamics of PKA signaling. While we showed previously that the RIIß holoenzyme could undergo a single turnover autophosphorylation with adenosine triphosphate and magnesium (MgATP) and trap both products in the crystal lattice, we asked here whether calcium could trap an ATP:RIIß holoenzyme since the RIIß holoenzyme is located close to ion channels. The 2.8Å structure of an RIIßp2:C2:(Ca2ADP)2 holoenzyme, supported by biochemical and biophysical data, reveals a trapped single phosphorylation event similar to MgATP. Thus, calcium can mediate a single turnover event with either ATP or adenosine-5'-(ß,γ-imido)triphosphate (AMP-PNP), even though it cannot support steady-state catalysis efficiently. The holoenzyme serves as a "product trap" because of the slow off-rate of the pRIIß subunit, which is controlled by cAMP, not by phosphorylation of the inhibitor site. By quantitatively defining the RIIß signaling cycle, we show that release of pRIIß in the presence of cAMP is reduced by calcium, whereas autophosphorylation at the phosphorylation site (P-site) inhibits holoenzyme reassociation with the catalytic subunit. Adding a single phosphoryl group to the preformed RIIß holoenzyme thus creates a signaling cycle in which phosphatases become an essential partner. This previously unappreciated molecular mechanism is an integral part of PKA signaling for type II holoenzymes.

Kinase regulation by hydrophobic spine assembly in cancer.

A new model of kinase regulation based on the assembly of hydrophobic spines has been proposed. Changes in their positions can explain the mechanism of kinase activation. Here, we examined mutations in human cancer for clues about the regulation of the hydrophobic spines by focusing initially on mutations to Phe. We identified a selected number of Phe mutations in a small group of kinases that included BRAF, ABL1, and the epidermal growth factor receptor. Testing some of these mutations in BRAF, we found that one of the mutations impaired ATP binding and catalytic activity but promoted noncatalytic allosteric functions. Other Phe mutations functioned to promote constitutive catalytic activity. One of these mutations revealed a previously underappreciated hydrophobic surface that functions to position the dynamic regulatory αC-helix. This supports the key role of the C-helix as a signal integration motif for coordinating multiple elements of the kinase to create an active conformation. The importance of the hydrophobic space around the αC-helix was further tested by studying a V600F mutant, which was constitutively active in the absence of the negative charge that is associated with the common V600E mutation. Many hydrophobic mutations strategically localized along the C-helix can thus drive kinase activation.

Kinases and pseudokinases: lessons from RAF.

Protein kinases are thought to mediate their biological effects through their catalytic activity. The large number of pseudokinases in the kinome and an increasing appreciation that they have critical roles in signaling pathways, however, suggest that catalyzing protein phosphorylation may not be the only function of protein kinases. Using the principle of hydrophobic spine assembly, we interpret how kinases are capable of performing a dual function in signaling. Its first role is that of a signaling enzyme (classical kinases; canonical), while its second role is that of an allosteric activator of other kinases or as a scaffold protein for signaling in a manner that is independent of phosphoryl transfer (classical pseudokinases; noncanonical). As the hydrophobic spines are a conserved feature of the kinase domain itself, all kinases carry an inherent potential to play both roles in signaling. This review focuses on the recent lessons from the RAF kinases that effectively toggle between these roles and can be "frozen" by introducing mutations at their hydrophobic spines.